To investigate the involvement of intrinsic mitochondrial apoptosis in dental monomer-induced cytotoxicity and the influences of N-acetyl cysteine (NAC) on this process.Human dental pulp cells (hDPCs) were exposed to several dental monomers in the absence or presence of NAC, and cell viability, intracellular redox balance, morphology and function of mitochondria and key indicators of intrinsic mitochondrial apoptosis were evaluated using various commercial kits.Dental monomers exerted dose-dependent cytotoxic effects on hDPCs. Concomitant to the over-production of reactive oxygen species (ROS) and depletion of glutathione (GSH), differential changes in activities of superoxide dismutase, glutathione peroxidase, and catalase were detected. Apoptosis, as indicated by positive Annexin V/propidium iodide (PI) staining and activation of caspase-3, was observed after dental monomer treatment. Dental monomers impaired the morphology and function of mitochondria, and induced intrinsic mitochondrial apoptosis in hDPCs via up-regulation of p53, Bax and cleaved caspase-3, and down-regulation of Bcl-2. NAC restored cell viability, relieved oxidative stress and blocked the apoptotic effects of dental monomers.Dental monomers induced oxidative stress and mitochondrial intrinsic apoptosis in hDPCs. NAC could reduce the oxidative stress and thus protect hDPCs against dental monomer-induced apoptosis. Owing to their physical and aesthetic properties, resin-based materials are routinely used to restore the structure and function of teeth , 2. HoweNovel strategies are needed to reduce the adverse effects of dental materials. To design such strategies, it is necessary to understand the exact mechanisms by which these materials induce cell death and to find strategies to decrease or eliminate their toxicities. N-acetyl cysteine (NAC) has played a well-documented role in detoxifying dental monomers and resinous materials –12. Howe2 at 37°C.Human dental pulp cells (hDPCs) were derived from primary culture, as described in our previous study . Briefly3 cells/well and incubated at 37°C and 5% CO2 for approximately 24 h. When the cells reached 80% confluence, media were removed. Cells were treated with media containing 2-hydroxyethyl methacrylate , methyl methacrylate , or triethylenglycol dimethacrylate  for 24 h. All the tested dental monomers were purchased from Sigma-Aldrich  and dissolved in complete media. CCK-8 solution was added, and the cells were incubated at 37°C for another 4 h. Absorbance of the colored solution was measured with a microplate reader  at a wavelength of 450 nm. Cells in the control group were treated with culture medium without dental monomers. Here, we found the lowest concentration for each monomer that could induce a significant decrease in cell viability . In the following study evaluating dental monomer-induced oxidative stress and apoptosis, 1 mM HEMA, 5 mM MMA and 1 mM TEGDMA were used.Firstly, Cell Counting Kit-8 (CCK-8)  was used to determine the influences of dental monomers on the viability of hDPCs. Briefly, hDPCs at the second passage were seeded into 96-well culture plates at 5 × 10Human DPCs were also exposed to 1mM HEMA, 5mM MMA and 1mM TEGDMA in the absence or presence of 10 mM NAC for 24, 48, 72 or 96 h to test the influence of NAC on monomer-induced cytotoxicity. NAC  was dissolved in complete media to a final concentration of 10 mM as previously described . After cLevels of intracellular ROS were measured with an ROS assay kit . After treatment with dental monomers  in the absence or presence of 10 mM NAC for 6 h, hDPCs were treated with dihydrodichlorofluorescein diacetate (DCF-DA), incubated in serum-free media at 37°C for 20 min, harvested in phosphate-buffered saline (PBS), and analyzed in a FACSCalibur flow cytometer (BD Biosciences). Results were plotted as the mean ± SD of three determinations per sample for each experiment, with 10,000 cells being analyzed for each sample.5 cells/well, incubated for 24 h, and treated with dental monomers  in the absence or presence of 10 mM NAC. After 24 h, the cells were collected and resuspended in the buffer for lyticase  for 30 min at 30°C. Then the lysate was centrifugated , and the supernatant was collected. Total protein concentrations of parallel samples were measured using a BCA Protein Assay Kit . The supernatant was aliquoted and stored at -80°C. All experiments were repeated three times.Human DPCs were seeded into a 6-well plate at a density of 1 × 10Contents of total glutathione , reduced glutathione (GSH) and oxidized disulfide (GSSG) were measured using a commercially available kit  according to the manufacturer’s protocol.After the supernatant was collected, total GSH was assayed using the 5,5-dithio-bis(2-nitrobenzoic) acid (DTNB)-GSSG reductase recycling. GSSG was measured by measuring 5-thio-2-nitrobenzoic acid (TNB) which was produced from the reaction of reduced GSH with DTNB. Then the fluorescence of the samples was evaluated using a microplate reader  at 405 nm. Total GSH and GSSG concentration were normalized to protein contents. The concentration of reduced GSH in the sample was obtained by subtracting GSSG from total GSH. The experiment was done in triplicate and repeated for three times.Lipid peroxidation was assayed by the measurement of malondialdehyde (MDA), according to the instructions of lipid peroxidation MDA assay kit . One hundred μL supernatant was mixed with 100 μL MDA working solution and boiled at 100°C for 15 min. Samples were cooled down to room temperature in a water bath and then centrifuged at 1,000g for 10 min. The absorbance at 532 nm was measured in a 96-well plate (200 μL/well) with a microplate reader . The results of MDA assay were expressed as micromoles of MDA per milligram of protein (nmol/mg protein). The experiment was done in triplicate and repeated for three times.The superoxide dismutase (SOD) activity was quantified with the SOD Assay Kit . The 50% inhibitory activity of SOD was determined by the colorimetric method and quantified by measuring the absorbance at 450 nm using a microplate reader . The activity of glutathione peroxidase (GPx) was quantified in the supernatant by using a GPx Assay Kit  and measuring the degree of reducing absorption of NADPH at 340 nm. The catalase (CAT) activity was quantified by using the CAT Assay Kit . The activities of SOD, GPx and CAT were normalized to protein contents. Results were plotted as the mean ± SD of three independent experiments with three determinations per sample for each experiment.5 cells/well, incubated for 24 h, and treated with HEMA (0-1-5-10 mM), MMA (0-5-10-15 mM) or TEGDMA (0-1-5-10 mM), in the absence or presence of 10 mM NAC. Untreated cells served as the negative control. After culturing for 24 h, hDPCs were collected and washed with PBS, gently resuspended in Annexin V binding buffer, and incubated with Annexin V-FITC/propidium iodide (PI). Flow cytometry was performed using flow cytometric analysis (Becton-Dickinson FACScan). Results were plotted as the mean ± SD of three determinations per sample for each experiment, and 10,000 cells were analyzed for each sample.Dental monomer-induced cell apoptosis and the protective effects of NAC were quantified with an Annexin V-FITC apoptosis detection kit . Human DPCs were seeded into a 6-well plate at 1×106 cells treated with dental monomers  with or without 10 mM NAC for 24 h were collected and lysed in a lysis buffer. Cell lysates were tested for protease activity by using Asp-Glu-Val-Asp-pNA (DEVD-pNA), a tetrapeptide p-nitroanilide substrate. Triple wells were used for each group. After incubation with this substrate for 2 h, the absorbance was measured at 405 nm with a microplate reader . Caspase enzymatic activities in cell lysates were normalized to protein contents measured in a BCA Protein Assay Kit. The results were presented as means ± SD of three independent experiments.To investigate caspase-3 activation after treatment with dental monomers in the absence or presence of NAC, the Caspase-3 Colorimetric Assay Kit  was used. Briefly, 1×105 cells/well. Then the cells were treated with dental monomers  in the absence or presence of 10 mM NAC for 24 h. After rinsed with PBS, the cells were schizolysised by solution and then centrifuged at 12,000g at 4°C for 15 min and the supernatant was collected. After 100 μL of the supernatant was mixed with 100 μL of ATP detection solution, intensity readings were taken with a microplate reader . The ATP concentrations in samples were calculated using an ATP standard curve and the protein concentration of each treatment group was determined using the BCA protein assay kit. The cellular ATP levels were expressed as nmol/mg protein.ATP level was measured by using a firefly luciferase based ATP Assay Kit  according to the manufacturer’s instructions. Cells were seeded into a 6-well plate at a density of 1×10The fluorescent dye JC-1  was used to detect cells with collapsed MMP. Briefly, cells cultured in α-MEM were exposed to dental monomers  in the absence or presence of 10 mM NAC for 24 h and incubated with the JC-1 staining solution (5 mg/ml) for 20 min at 37°C. Cells were rinsed twice with JC-1 staining buffer. The fluorescence intensity (FI) of JC-1 aggregates was detected at an excitation/emission wavelength ratio of 525 nm/590 nm, and the FI of the JC-1 monomers was measured at an excitation/emission wavelength ratio of 490 nm/530 nm by flow cytometry (Becton-Dickinson FACScan). All results were plotted as the mean ± SD of three determinations per sample for each experiment, and 10,000 cells were analyzed for each sample.n = 3) and lysed in RIPA buffer. Protein contents of the lysed cells were measured with the BCA Protein Assay Kit. Extracted proteins were loaded on 10% sodium dodecyl sulfate polyacrylamide gels, transferred to polyvinylidene fluoride membranes (Bio-Rad), and blocked with 5% nonfat milk powder. Membranes were incubated overnight with the following primary rabbit anti-human antibodies : anti-Bcl-2, anti-Bax, anti-p53, anti-cleaved caspase-3, and actin. Membranes were incubated with goat anti-rabbit secondary antibodies. Protein signals were visualized by using the ECL Western Blotting Detection System . Protein expression levels were normalized to actin by using Image-Pro Plus 5.0 Software .Human DPCs cultured in α-MEM were exposed to dental monomers  in the absence or presence of 10 mM NAC for 24 h  without or with 10 mM NAC for 24 h, the release of CytoC from mitochondria to the cytoplasm was investigated. Cells were resuspended in 1.5 mL of cold Mito-Cyto Buffer , lysed, and centrifuged. The supernatant was centrifuged again at 12,000 g for 10 min to separate mitochondria (in pellet) from the cytoplasm (in supernatant). Cytosolic Cyto C in the supernatant was quantified with an ELISA kit , using Cyto C (0–2000 pg/mL) as a standard. Data are expressed as the total amount of intracellular solubilized Cyto C. Cyto C levels were normalized to protein content measured by a BCA Protein Assay Kit. All results were plotted as the mean ± SD of three determinations per sample for each experiment.5 cells/well and exposed to dental monomers  without or with 10 mM NAC for 24 h. The cells were then fixed with a modified Zamboni's fixative  for 30 min at room temperature as described before [Human DPCs were seeded into a 6-well plate at a density of 1×10d before . Cells wHuman DPCs were initially collected after exposure to dental monomers  without or with 10 mM NAC for 24 h, and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 h and post fixed with a solution of 1% osmium tetroxide in 0.1 M cacodylate buffer. The cells were then embedded in epoxy resins after a graded-ethanol serial dehydration step. The embedded cells were sectioned into ultrathin slices, stained by uranyl acetate solution and lead citrate, and then observed with a transmission electron microscope Tecnai G2 Spirit BioTWIN electron microscope .P = 0.05 for all tests.The SPSS 18.0 software package  was used to perform statistical analyses. Data was analyzed by one-way analysis of variance (ANOVA), followed by the Tukey’s test. The statistical significance level was set at P < 0.05 as compared to control group). Likewise, similar time-dependent inhibitory effects were observed in cells exposed to 5 mM MMA and 1 mM TEDGMA . Quite the reverse, the presence of NAC could significantly restore the cell viability  for all the studied time periods.The CCK-8 assay results revealed that treatment of hDPCs with dental monomers for 24 h decreased cell viability in a dose-dependent manner . CompareP < 0.05, P < 0.05).Compared to the control group, the intracellular ROS levels were increased in hDPCs after treatment with dental monomers for 6 h . Compared to the control group, no difference in GSSG content was found after treatment with HEMA or MMA .MDA level was remarkably increased after dental monomer treatment . For MMA and TEGDMA, NAC could also alleviate decreased activity of SOD, although there was no significant difference . Similar results were observed for GPx activity .SOD activity decreased after dental monomer treatment (activity . CAT actP > 0.05 as compared to the control group). Dental monomers greatly dose-dependently increased the percentage of cells in early apoptosis as well as in late apoptosis/necrosis. Quite the reverse, NAC exhibited restoring effects on cell viability and meanwhile decreased the percent of early apoptotic and late apoptotic/necrotic cells. Compared to untreated cells, there was no statistical difference in late apoptosis/necrosis in 1 mM HEMA, 5 mM MMA and 1 mM TEGDMA-treated cells in the presence of NAC (P > 0.05 as compared to the control group).According to the results of Annexin V/PI staining, a dose-dependent decrease in the percentage of viable cells was observed in hDPCs exposed to dental monomers . It was P < 0.05 vs. control group cells). In contrast, co-treatment with NAC significantly inhibited dental monomer-induced activation of caspase-3 .The occurrence of apoptosis was also confirmed by evaluating the activity of caspase-3, whose activation plays a central role in the execution-phase of cell apoptosis , 17. As P< 0.05, P> 0.05 as compared to the control group).Cellular ATP levels of hDPCs exposed to dental monomers, in the absence or presence of NAC, were demonstrated in P < 0.05 vs. control group cells). NAC protected against dental monomer-induced depolarization of MMP .Next, we analyzed the effects of dental monomers and NAC on MMP, which was an important biomarker of intrinsic mitochondrial pathway. As can be seen in The morphologies of mitochondria in cells exposed to dental monomers, in the absence or presence of NAC, were examined by transmission electron microscope (TEM) here. After monomer treatment, some cells showed nuclear and cytoplasmic signs of apoptosis . As for the mitochondira, the control cells  and NAC-P < 0.05 as compared to the control group, P < 0.05 for TEGDMA as compared to the control group). The expression levels of Bax after HEMA or MMA treatment also showed a trend of increase, although there was no statistical significance compared to the control group . Co-treatment with NAC resulted in enhanced Bcl-2 expression , and reduced Bax expression . p53 is a sensor of cellular stress and is a critical activator of the intrinsic mitochondrial apoptosis pathway. The p53-dependent regulation of apoptosis occurs through transcription dependent and transcription-independent mechanisms to either activate or in activate multidomain pro- and anti-apoptotic Bcl-2 proteins [P < 0.05 vs. control group cells) and NAC co-treatment significantly reversed dental monomer-induced up-regulation of p53. As for caspase-3, which is the executor of apoptosis, dental monomers greatly enhanced the intracellular level of cleaved caspase-3 .Bcl-2 protein family is the key regulator of intrinsic mitochondrial apoptosis. Among the many members of Bcl-2 family proteins, Bcl-2 and Bax are two well-known molecules with anti-apoptotic, or proapoptotic effects, respectively , 18. Wesproteins . Dental P < 0.05 vs. control group cells, P < 0.05 vs. dental monomer-treated cells). These observations indicate that NAC prevented the release of Cyto C from mitochondria into the cytoplasm after dental monomer treatment.Cyto C is an important intermediate in intrinsic mitochondrial apoptosis. The release of Cyto C from mitochondria could activate caspase-9, which in turn activates caspase-3 and caspase-7 to execute cell death , 20. ResImmunofluorescence (IF) analysis revealed that Bax, which is diffusely present as fine granules in the cytosol of controls, translocated to mitochondria after dental monomer treatment with a punctate perinuclear distribution typical of mitochondria . In contIn the present study, we demonstrated that dental monomers could disturb the intracellular redox balance, impair the functions of mitochondria and thus induced apoptosis through the intrinsic mitochondrial pathway in hDPCs. The presence of NAC could relieve dental monomer-induced oxidative stress and subsequently protect the cells from apoptosis.In our study, the results of CCK-8 assay revealed that dental monomers dose-dependently inhibited cell viability of hDPCs. However, the tested monomers revealed different level of toxic effects on hDPCs, with MMA showing the lowest toxicity, followed by HEMA and then TEGDMA. This result is in accordance with previous studies , 22. TheThe dental monomer-induced cytotoxicity was paralleled by over-production of ROS and depletion of GSH in hDPCs. This result is in accordance with previous studies, which indicate that oxidative stress is the primary mechanism for the toxic effects of dental monomers , 27. GSS2- into O2 and H2O2, whereas CAT and GPx subsequently conduct the conversion of H2O2 to H2O and O2 [2O2 was the major sort of ROS after dental monomer treatment [2O2, the limited source of the substrate GSH as a consequence of monomer-induced GSH depletion can subsequently restrict the activity of GPx, causing the observed reduction in GPx activity [2O2 formation may lead the increased CAT activity and decreased SOD activity levels as a result of a feedback regulation [Besides the non-enzymatic antioxidant GSH, enzymatic antioxidants directly control cellular redox homeostasis by regulating the levels of particular ROS. In this study, CAT activity was increased after exposure to dental monomers, whereas SOD and GPx activities were reduced. Functionally, SOD catalyzes the breakdown of OO and O2 , 33. A rreatment . Becausegulation .In this study, a dose-dependent decrease in the percentage of viable cells, as well as an increase in apoptotic and necrotic cells was observed in hDPCs exposed to dental monomers. Furthermore, dental monomers enhanced the activity of caspase-3. These data is in accordance with many previously published researches . AlthougBased on these results, we then moved on to study the expression level of other key molecules of the intrinsic mitochondrial apoptosis pathway to support our hypothesis. The results of Western Blot analysis revealed that dental monomers induced upregulation of the pro-apoptotic protein Bax and cleaved caspase-3, as well as the downregulation of the anti-apoptotic protein Bcl-2. The intrinsic mitochondrial apoptosis pathway had been well studied by numerous studies , 37. ApoNAC is a well-known free radical scavenger that can readily enter the cells. We found that NAC could alleviate dental monomer-induced oxidative stress and thus protected the cells from apoptosis. The protective effects of NAC may be attributed to three aspects. First, it can directly scavenge the over-produced ROS. Second, as a cysteine-donating compound, NAC acts as a cellular precursor of GSH to replenish the depleted GSH pool after dental monomer treatment . Third, in vitro study, it might be concluded that dental monomers caused disturbance of intracellular redox balance, which is characterized by depletion of GSH, over-production of ROS, and differential changes of antioxidative enzymes. The oxidative stress subsequently impaired mitochondria function, up-regulated p53, and eventually initiated the intrinsic mitochondrial apoptosis pathway. NAC remarkably relieved dental monomer-induced oxidative stress and subsequently protected the cells against apoptosis.The conclusions from the present findings are summarized in a hypothetical model . With thS1 Raw Data(XLS)Click here for additional data file.


Moreover, the FO performance of the resultant membrane was measured by using 2 M MgCl2 solution as draw solution and deionized (DI) water as feed solution, respectively. The membrane with a protein-to-lipid weight ratio (P/L) of 1/50 exhibits 13.2 L/m2h water flux and 3.2 g/m2h reversed flux by using FO mode, as well as 15.6 L/m2h water flux and 3.4 L/m2h reversed flux for PRO mode (the draw solution is placed against the active layer). It was also shown that the SLB layer of the double-skinned FO membrane can increase the surface hydrophilicity and reduce the surface roughness, which leads to an improved anti-fouling performance against humic acid foulant. The current work introduced a new method of fabricating high performance biomimetic FO membrane by combining AqpZ and a double-skinned structure based on LbL assembly.We demonstrated a novel AquaporinZ (AqpZ)-incorporated double-skinned forward osmosis (FO) membrane by layer-by-layer (LbL) assembly strategy. Positively charged poly(ethyleneimine) (PEI) and negatively charged poly(sodium 4-styrenesulfonate) (PSS) were alternately deposited on both the top and bottom surfaces of a hydrolyzed polyacrylonitrile (H-PAN) substrate. Subsequently, an AqpZ-embedded 1,2-dioleloyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-3-trimethylammonium- propane  (DOTAP) supported lipid bilayer (SLB) was formed on PSS-terminated (T-PSS) membrane via vesicle rupture method. The morphology and structure of the biomimetic membranes were characterized by  It has et al. reported on an AqpZ-embedded supported lipid bilayer (SLB) by using an electrostatic layer-by-layer (LbL) deposition method to prepare a robust biomimetic membrane, which demonstrated an excellent nanofiltration (NF) performance [et al. fabricated SLB biomimetic membranes by spin-coating positively charged proteoliposomes on a modified NF-270 membrane via vesicle fusion [et al. encapsulated magnetic nanoparticles into the vesicles and utilized magnetic force to enhance the adsorption amount of AqpZ-incorporated vesicles on the polyelectrolyte film [et al. reported a method of synthesizing a FO biomimetic membrane [To date, several methods have been applied to fabricate AQP-containing biomimetic membranes, most of which are based on depositing AQP-incorporated vesicles onto porous substrates. Many efforts have been made to improve the performance of the AQP-based biomimetic membranes, including direct deposition of vesicles onto the substrates, pressure-assisted vesicle fusion, electrostatic force-induced vesicle adsorption, magnetic-enhanced vesicle deposition, and chemical crosslinking between functionalized lipid and the substrate ,5,6,7. Wformance . Li et aetc. [Forward osmosis (FO) utilizes an osmotic pressure gradient across the semipermeable membrane to separate clean water from feed solution (FS) . Advantaetc. ,10,11. Hetc. ,13. In tetc. ,15. Howeetc. . In ordeetc. ,18. Competc. .In our work, we combined AqpZ with double-skinned FO membranes to further improve the performances of the biomimetic FO membranes. The electrostatic LbL deposition method, which is based on the alternating adsorption of oppositely charged components ,21, has N,N-dimethylformamide (DMF) and lithium chloride were purchased from Sinopharm . Polyethyleneimine  and poly(sodium 4-styrene-sulfonate) , 1-n-octyl-β-d-glucopyranoside  and glycerol (purity > 99%) were purchased from Sigma-Aldrich . 1,2-dioleloyl-sn-glycero-3-phosphocholine  and 1,2-dioleoyl-3-trimethylammo-nium-propane  (DOTAP) were obtained from Avanti Polar Lipids (USA). AqpZ was expressed and purified using previously described procedures [2PO4, 10 mM K2HPO4 3H2O, and 150 mM NaCl.Polyacrylonitrile  was kindly supplied by the Shanghai Jingshan Petrochemical Company. ocedures . Deioniz2 stream was used to evaporate chloroform from a chloroform solution of DOPC/DOTAP mixture with a molar ratio of DOPC: DOTAP = 4:1 to obtain a lipid film. The dried lipid film was left standing overnight in a vacuum to remove the residual organic solvent, and then rehydrated in phosphate buffer through vortexing, followed by five freeze-thaw cycles. The final solution was extruded 11 times with 100 nm polycarbonate membranes through an extruder system  to get the uniform small unilamellar vesicles with a final concentration of 0.1 mg/mL.Liposome samples were prepared by the film rehydration method as previously described . BrieflyA certain amount of AqpZ solution was added into 0.1 mg/mL liposome solution containing 1 wt % OG to prepared samples with different protein-to-lipid ratios . The solution was dialyzed in a 6–8 KDa molecular weight cut-off (MWCO) dialysis bag (Spectra Por) against 1 L phosphate buffer for three days at room temperature. The buffer was refreshed every day. After dialysis, the proteoliposome solution was extruded through a polycarbonate membrane with a mean pore size of 100 nm as stated above for further use.PAN substrate was fabricated by using the phase inversion method. The PAN casting solution consisted of 18 wt % PAN and 2 wt % LiCl in DMF. The solution was stirred for 24 h at 60 °C to obtain a homogeneous solution and then cooled to room temperature for 24 h to remove the residual bubbles. A casting knife was used to cast the homogeneous solution on a clean glass plate, and then the glass plate was immediately immersed into a distilled water coagulation bath to form PAN substrates. Subsequently, the PAN substrates were soaked in a 1.5 M NaOH solution at 45 °C for 1.5 h and then rinsed by DI water until the pH of the rinse water became neutral [The LbL membrane was prepared by immersing the H-PAN substrate in 1 wt % PEI and 1 wt % PSS solution alternatively. Each deposition step lasted 30 min followed by a rinse of DI water for 5 min to remove the redundant polyelectrolytes. The procedure above could be repeated so that multilayer polyelectrolyte membranes with a specific bilayer number were fabricated. In our work, we adopted one PEI/PSS layer for the top rejection skin and then a similar method was used for the preparation of the bottom rejection layer. In that way, T-PSS membranes were fabricated.The prepared liposome or proteoliposome solutions were spread on the top layer of the T-PSS membrane and incubated at room temperature for 4 h. Then the biomimetic membrane was rinsed by DI water for 5 min to remove the residual vesicles.∆osm = 290 mosm/L, Osmomat 030, Gonotec, Germany) impelled the water effluent from the vesicles. By fitting the data to an exponential increase equation, the initial rate of vesicle shrinkage (k) was determined. Thereafter, the osmotic water permeability (fP) was calculated by using the following equation:S/V0 is the vesicle’s initial surface-to-volume ratio, VW is the molar volume of water (0.018 L/mol), and ∆osm is the osmolarity difference after mixing. All of the measurements were carried out at 25 ± 0.1 °C.Zetasizer  was used to characterize the size and zeta potential of liposomes and proteoliposomes. A 633 nm laser source, with a fixed detector angle of 90°, was used in the dynamic mode. We used a stopped-flow apparatus , with light source at emission wavelength of 577 nm, to characterize the permeability of the liposomes and proteoliposomes. Samples were rapidly mixed with a high-osmotic solution (0.5 mol/L sucrose). The osmolarity difference  spectrometer , using the attenuated total reflection (ATR) technique.The hydrophilicity of the prepared membrane was characterized by an automatic contact angle meter . Then 0.5 mL DI water was dropped onto the membrane surface using a microsyringe under ambient conditions and contact angle was measured until no further change. At least six random locations were measured for each sample in order to minimize the experimental error and then obtain the average value. Both the top surfaces and bottom surfaces of the double-skinned LbL FO membranes were measured.The surface morphology and roughness of the membranes were investigated via atomic force microscopy . Tapping mode measurements in liquid were performed by using NP-S cantilevers . Surface roughness values were obtained from AFM images with the help of the instrument software.The morphology and structure of original and modified membranes were analyzed by scanning electron microscope . Dry membrane samples were frozen in liquid nitrogen and subsequently cracked in order to obtain the cross sections.2. The linear velocity was 6.4 cm/s. We denoted the AqpZ-embedded SLB active layer facing FS as the FO mode. Accordingly, the active layer facing DS was denoted as pressure retarded osmosis (PRO) mode. The FO performances of double-skinned membranes were evaluated under both FO and PRO mode. As a control, single-skinned membranes, which were prepared by depositing only the top layer of H-PAN with polyelectrolytes and AqpZ-embedded SLB, were also measured.FO performances of the fabricated membranes were conducted in a lab-scale cross-flow FO filtration unit with an effective membrane area of 36.0 cm2 solution was used as DS and DI water was used as FS, respectively. The water flux was calculated by measuring the weight change of DS every 2 min. The solute reverse flux of the membrane was calculated from the conductivity change.Then, 2 mol/L MgClJv in L/m2·h) and solute reverse flux (Js in g/m2·h) were calculated by the following equations [V is the volume changes of DI water (density is 1000 g/L); Am is the effective membrane area; Δt is the measuring time interval.V0 and tV are the initial and final volumes of feed solution, respectively; C0 and tC are the initial and final solute concentrations of feed solution, respectively.The water flux J2 DS and DI water FS for 1 h to obtain the initial water flux. Anti-fouling tests were performed with 5 mg/L humic acid (HA) as the FS for 1 h. Subsequently, both the FS and DS were changed into DI water to rinse the membrane for 0.5 h, which was the cleaning process. Finally, 2 mol/L MgCl2 DS and DI water FS were used for 1 h to measure the recovered water flux. The fouling and cleaning cycles were performed three times, alternatively.The anti-fouling tests of double-skinned membranes were performed in the FO mode. Prior to the fouling test, the FO membrane was first tested using 2 mol/L MgClk) than liposomes, which could be attributed to the higher water permeation of AqpZ. Moreover, the permeability of proteoliposomes increased as the P/L ratio increased because more incorporated AqpZ enhanced the permeability of the vesicles. Based on the data of the proteoliposomes with the protein-to-lipid molar ratio of 1:50, the permeability of each AqpZ was calculated to be 32.4 × 10−14 cm3·s−1, which was comparable to the previously reported results [ results ,31.−1 was due to stretching vibration of the C≡N moiety of the PAN substrate. After being treated by 1.5 M NaOH solution, most of the CN groups convert to COO− groups as previously reported [−1, representing the C=O bond in COO− groups. After PEI deposition, the intensity of the N–H bend and C–N stretch of the CONH group increased at around 1553 cm−1, which suggested that the nucleophilic amine groups were formed on the membrane. For the T-PSS membrane, two new peaks appeared at 1068 cm−1 and 1042 cm−1, which corresponded to the S=O bond stretching vibration. When AqpZ-incorporated DOPC/DOTAP SLB was formed on the polyelectrolyte membrane (the membrane we denoted as T-AqpZ), the characteristic peak attributed to C–C–N+ groups appeared at around 947 cm−1. As well, the peak at 715 cm−1 was attributed to the vibration of P–O–C groups. The spectra of the membrane bottom surfaces with different outermost layers were shown in ATR-FTIR analyses were performed to verify that the AqpZ-incorporated double-skinned FO membranes were successfully fabricated. As shown in reported . As a re− groups and some amine groups on the surface of the T-PSS membrane have a strong interaction with water molecules. Finally, after covering with proteoliposomes, the water contact angle made a further decrease to 20.1°. This can be understood by the fact that the SLB with high hydrophilicity covered the surface of the membrane and greatly improved the surface hydrophilicity [The hydrophilicity of the membrane surface is usually evaluated by the water contact angle. In general, a higher contact angle of the membrane surface indicates higher hydrophobicity . The wathilicity .-embedded SLB, the gaps in the top surface were filled so that the membrane appeared to be smoother and less defective. As for the cross-section images, finger-like pores were formed in the asymmetric membranes [The SEM images of fabricated membranes are shown in embranes . Both thembranes .AFM was used to characterize the roughness of various membranes. As shown in 2 solution as the DS and DI water as the FS. Both the FO and PRO modes (the draw solution is placed against the active layer) were measured and shown in Jv of AqpZ-absent SLB-covered double-skinned FO membrane was 3.5–4.8 L/m2·h and the Js/Jv was about 0.2 g/L. The low water flux could be explained by the fact that the lipid bilayer was impermeable to water. However, due to the existence of defects on the SLB, the membrane exhibited a low water flux and reversed salt solution flux [Jv increased from 13.7 to 15.6 L/m2·h by using the PRO mode, while the Js/Jv slightly increased from 0.21 to 0.24 g/L. This can be understood by considering the fact that plenty of water channels were provided by AqpZ for water molecules to pass through the biomimetic membranes. However, the zeta potential of proteoliposomes decreased with the increase of the P/L ratio , which confirmed the fact that AqpZ was beneficial to FO biomimetic membranes.As shown in 2 as the DS and DI water as the FS for 1 h. The water flux slightly dropped because of the decrease of the driven pressure induced by the dilutive ICP [The FO fouling tests were performed with AqpZ (P/L = 1/50)-incorporated membranes in the PRO mode. In the PRO mode, foulants could go into the porous support easily. Therefore, the PRO mode was used to test the anti-fouling capacity of the double-skinned FO membrane. tive ICP . Remarkative ICP ,17. ThisWe successfully fabricated and characterized the AqpZ-incorporated double-skinned FO membranes based on layer-by-layer assembly in the current work. To the best knowledge of the authors, this is the first study reporting the combination of AqpZ and double-skinned FO membranes. The addition of AqpZ has made a great contribution to the water flux and salt rejection. The changes were obvious in the water flux. The double-skinned FO membranes possessed a relatively loose secondary selective layer, which decreased ICP and ensured the water flux. Through fouling tests, the double-skinned FO membranes demonstrated a much better anti-fouling capacity compared to the single-skinned FO membranes.


The Obsessive Beliefs Questionnaire-44 (OBQ-44) is originally developed by the Obsessive Compulsive Cognitions Working Group and has been translated into several languages. This paper is aimed to investigate the psychometric properties of the Chinese version of the Obsessive Beliefs Questionnaire-44 (OBQ-44) in both clinical and non-clinical samples.Five hundred and sixty-nine undergraduate volunteers and sixty-six OCD patients were included in the study. All participants have completed Chinese version of OBQ-44, Yale-Brown Obsessive-Compulsive Scale (Y-BOCS), and Beck Depression Inventory (BDI). Confirmatory factor analysis was conducted to examine the construct validity of Chinese version of OBQ-44. The internal consistency and test-retest reliabilities at 4-week interval were examined in both non-clinical and clinical groups.χ2/d.f = 2.96, GFI = 0.83, NFI = 0.82, CFI = 0.88 and RMSEA = 0.06). The internal consistency and test-retest reliability were at an acceptable range for the two samples.The confirmatory factor analysis of the non-clinical sample confirmed a 3-factor model which was suggested by the original authors of the instrument (The Chinese version of OBQ-44 is a valid and reliable instrument for assessing dysfunctional beliefs related to the etiology and maintenance of obsessions and compulsions. Obsessive-compulsive disorder (OCD) is characterized by the presence of obsessions and/or compulsions that are time-consuming and usually provoke distress . A consiThus, the current study is aimed to investigate whether OBQ-44 is reliable and valid in Chinese population. We translated it into Chinese, and then we validate its psychometric properties in non-clinical and clinical samples. The availability of Chinese OBQ-44 would provide Chinese researchers with a valid measurement to evaluate the severity of dysfunctional beliefs in Chinese context, and to facilitate cross-cultural comparison in the future.The non-clinical sample was made up of five hundred and sixty-nine undergraduate volunteers recruited from Sun Yat-Sen University in Guangzhou, China . Theirs ages ranged from 17 to 23, and the average age was 19.79 (SD = 1.68). Sixty-six OCD patients were the out-patients of Guangzhou Psychiatry Hospital. Nineteen (28.8 %) of participants were female, and forty-seven (71.2 %) were male. Theirs ages ranged from 12 to 44, and the average age was 25.15 (SD = 7.98). The average years of their education was 12.56 (SD = 3.29). OCD patients were diagnosed with DSM-IV criteria for OCD by Structured Clinical Interview (SCID) [Potential non-clinical and clinical participants were excluded if they: (1) were younger than 18 or older than 50; (2) had a history of head injury, central nervous system diseases, or mental illness (except OCD patients); (3) had a history of substance abuse. These exclusion criteria ensured that all the participants could understand the procedures in the study.The present study was approved by the ethics committee of Guangzhou Psychiatry Hospital. Written consent was obtained from each study participant before the survey, and the confidentiality of the data use was ensured. Then the participants were instructed to complete the questionnaires. The non-clinical samples were undergraduate volunteers. They completed the questionnaires in the classroom at one time. The clinical samples were OCD patients and they completed the questionnaires in the hospital one by one. All research assistants were very well trained in administering the instruments before the survey.OBQ-44 is a self-reporting questionnaire including 44 items to evaluate the belief domains associated with OCD. The revised version has three subscales: responsibility/threat estimation (RT) with 16 items , importance/control of thoughts (ICT) with 16 items , and perfectionism/certainty (PC) with 12 items . The respondents are requested to score himself or herself what degree the situation described in each particular statement by a seven-point scale . The total score ranges from 44 to 308.The validation of the Chinese version of OBQ-44 followed the international guidelines suggested by Beaton for cross-cultural validation of self-reported measures: (1) the initial translation of the original scale into the used language; (2) the synthesis of conceptions; (3) the back-translation; (4) the expert committee’s review on the relevance and representation of items used for the final outcome setting, and (5) the piloting of testing and probing to get at understanding of item . After hThe Chinese version of Y-BOCS was used to assess the severity of OCD symptoms and to provide measurements of concurrent validity of the translated OBQ-44. Y-BOCS includes 10 items, scoring on a 5-point Likert scale ranging from 0 (disagree very much) to 4 (agree very much). The first five items are summed to provide an obsession severity score (OS) and the last five items shows the compulsions severity score (CS). The Chinese version of Y-BOCS has excellent convergent and divergent validity; the retest reliability and internal consistency are satisfactory . CronbacThe 21-item BDI was used in present study to assess the severity of depression symptoms over the past two weeks and to provide measurements for the concurrent validity of the translated OBQ-44. Responses were provided on 4-point Likert scale ranging from 0 (symptoms not present) to 3 (symptom strongly present). The Chinese version of BDI has excellent split reliability and the internal consistency is satisfactory . Cronbacχ2/d.f, Comparative Fit Index (CFI), Root Mean Square Error of Approximation (RMSEA), Goodness of Fit Index (GFI) and Normed Fit Index (NFI). We used the criteria recommended by Hu and Bentler [χ2/d.f ≤ 3, CFI ≥ 0.95, RMSEA ≤ 0.08, GFI ≥ 0.90 and NFI ≥ 0.90. The Cronbach’s alpha and student’s t-test for paired samples were used respectively to assess the internal consistency and the test-retest reliability. Moreover, the convergent validity of the Chinese version was also assessed by Pearson correlations with Y-BOCS and BDI respectively, and the comparison between the score of non-clinical sample and that of clinical sample was also conducted. Two-tailed significance level of 0.05 was considered for all statistical procedures in this study.Data were sorted and analyzed using SPSS 20.0 and AMOS 7.0.We performed a confirmatory factor analysis in the non-clinical sample to test three-factor structure of the Chinese version of OBQ-44 by AMOS 7.0. The goodness-of-fit indexes used in the present study as follow:  Bentler  for goodχ2/d.f = 2.96, GFI = 0.83, NFI = 0.82, CFI = 0.88, RMSEA = 0.06 and SRMR = 0.06. Figure We performed a CFA to test the applicability of OBQ-44 to Chinese culture. In our study, maximum likelihood estimation method on the covariance matrix was used to estimate the model fit. The results from CFA suggested an acceptable fit for the model: The internal consistency and test-retest reliability of the instrument were calculated respectively for the non-clinical group and clinical group (see Table n = 569), Cronbach’s alpha for overall scale and three subscales are listed as follows: overall OBQ-44, α = 0.954; RT, α = 0.902; PC, α = 0.889; ICT, α = 0.840. For the clinical group (n = 66), Cronbach’s alpha for overall scale and three subscales are listed as follows: overall OBQ-44, α = 0.962; RT, α = 0.930; PC, α = 0.917; ICT, α = 0.869. All revealed a very good consistency.In non-clinical group (n = 371), the coefficients of test-retest correlation are 0.709 for RT, 0.729 for PC, 0.635 for ICT, and 0.786 for the whole scale. Moreover, for the clinical sample (n = 23), the coefficients of test-retest correlation are 0.752 for RT, 0.745 for PC, 0.653 for ICT, and 0.723 for the scale. All test-retest correlations were significant, which indicates OBQ-44 has adequate levels of stability for a measurement.For the non-clinical sample (P < 0.01) in non-clinical sample, and 0.312 (P = 0.012 < 0.05) in clinical sample. These results showed the OBQ-44 had an acceptable convergent validity. Table The convergent validity of OBQ-44 was assessed by calculating the correlations between OBQ-44 scores  and the scores of Y-BOCS and BDI in both non-clinical sample and clinical sample Table . If the The purpose of this study was to examine the psychometric properties of Chinese version of OBQ-44 in both clinical and non-clinical samples. In our non-clinical sample, a confirmatory factor analysis confirmed the three-factor structure of the OBQ-44, namely responsibility/threat estimation (OBQ-RT), perfectionism/certainty (OBQ-PC), and importance/control of thoughts (OBQ-ICT). These results indicated that Chinese version of OBQ-44 is consistent to the model of the original version , BraziliAn analysis of the whole scale and the three subscales for non-clinical samples demonstrated the Chinese version of OBQ-44 have high internal consistencies as indicated by the Cronbach’s alpha coefficients, which is consistent with the results reported by Julien , and slin = 569) and clinical samples (n = 66). Although the Chinese version shows less test-retest reliabilities compared to that of the French version [For clinical samples, the Cronbach’s alpha coefficients of 44 items and three subscales showed a very good internal consistency for the Chinese version of OBQ-44, which is consistent with those reported by Bortoncello . Moreove version , the tes version .et al. [As expected, zero-order correlations between the OBQ-44 scores  and the other clinical measures i.e. Y-BOCS, and BDI were significant. There were partial support for the correlations between the OBQ-44 scores  and the Y-BOCS were significant and the correlations showed a tendency to be stronger than the correlation between the OBQ-44 scores  and BDI in non-clinical sample. However, in the clinical sample the correlations between the OBQ-44 scores  and the Y-BOCS showing a tendency to be lower than the correlation between the OBQ-44 scores  and BDI. Julien et al.  also repA major strength of the present study is that non-clinical and clinical samples were used to the validation of the Chinese version of OBQ-44. However, there are several limitations to be considered. First, the clinical sample is relatively small, and all the participants in the non-clinical sample were undergraduates, and female participants were over represented in the non-clinical sample, which may limit the generalizability of our finding. Psychometric analyses should be carried out in a more large and representative sample. Moreover, because the divergent validity of the Chinese version was only determined by the comparison of the OBQ-44 score of the OCD patients and of the undergraduates, and did not assess the anxiety level to show divergent validity. This is another limitation of this study. Finally, more information about the Chinese version’s the correlations with multiple scales of OCD symptoms should be used to examine its convergent validity.The results of the current study suggest that the Chinese version of OBQ-44 is a reliable and valid instrument to assess dysfunction beliefs in Chinese population. It is also useful for researchers to evaluate interventions aiming to reduce dysfunction beliefs. Furthermore, its validity is contributed to the cross-cultural comparison in the future.


However, there are particular pancreatic tumors having various grades of malignancy such as intraductal papillary mucinous neoplasm (IPMN) or pancreatic neuroendocrine tumor. We examined whether the cut-off value of maximum standardized uptake value (SUVmax) determined by pancreatic ductal cancers is also applicable for other pancreatic tumors.Positron emission tomography with 2-deoxy-2-[One hundred thirty six patients with pancreatic tumors underwent FDG-PET imaging. We first analyzed the cut-off value to differentiate pancreatic ductal cancers from mass-forming chronic pancreatitis. Secondly, we determined the cut-off value between malignant IPMN and benign IPMN. Thirdly, we computed a cut-off value between malignant pancreatic tumors and benign tumors irrespective of tumor type.The optimal cut-off value to differentiate ductal cancers from mass-forming chronic pancreatitis was 2.5. The optimal cut-off value for differentiating malignant IPMN from benign IPMN was also 2.5, similar to that of reported studies. In all types of pancreatic tumors, the cut-off value was also 2.5. The accuracy for detecting malignancy was 93.4% for all tumors.max of 2.5 would be justified as a cut-off value to differentiate malignant lesions.In the FDG-PET study for pancreatic tumors, an SUV Owing the progress of imaging modalities such as abdominal ultrasonography (US) and computed tomography (CT), accuracy in the diagnosis of pancreatic tumors has improved over the last decade. In pancreatic tumors, however, there are a variety of tumors such as ductal neoplasms, inflammatory and fibrotic tumors, and cystic tumors with malignant potentials of various degrees. Among them, intraductal papillary mucinous neoplasm (IPMN) and pancreatic neuroendocrine tumor (pNET) are the tumors in which it is difficult to distinguish malignant ones from benign ones preoperatively.18F]fluoro-D-glucose (FDG-PET) is a noninvasive, useful imaging modality. Theoretical background is based on the difference of cellular glucose metabolism  is a common parameter for evaluating the uptake by a mass lesion semi-quantitatively. This value is defined as the radioactivity of the tissue divided by the total radioactivity of the probe isotope injected per body weight . Figure max to differentiate ductal cancer from mass-forming chronic pancreatitis was 2.5, which was determined as the point of the curve farthest from the chance line. The areas divided by the curve were used to verify the performance of the analysis. The area under the curve was 0.982, which showed this analysis was appropriate. When an SUVmax of 2.5 was set as the cut-off value, only 4 of the ductal cancer lesions  had an SUVmax below the cut-off value . On the other hand, only one mass-forming chronic pancreatitis lesion (SUVmax = 5.3) had an SUVmax above the cut-off value . This patient has IgG4-related autoimmune pancreatitis (AIP). To summarize, when the cut-off value was set at 2.5, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy rate between ductal cancer and mass-forming chronic pancreatitis were shown in Table Table max in malignant IPMN (IPMC) and benign IPMN are illustrated in Figure P < 0.01). Figure max to differentiate IPMC from benign IPMN was also 2.5. The area under the curve was 0.933. When an SUVmax of 2.5 was set as the cut-off value, 4 of the malignant IPMNs  showed an SUVmax below the cut-off value . These 4 patients were all carcinoma in situ (CIS). There was no patient showing false-positive in the benign IPMN group.The SUVmax in all malignant tumors and all benign tumors was illustrated in Figure P < 0.01). The optimal SUVmax to differentiate all malignant tumor lesions from all benign tumor lesions was exactly 2.5  had an SUVmax below the cut-off value . On the other hand, in all benign tumors, only one patient (IgG4-related AIP) showed false-positive. Sensitivity, specificity and accuracy rate were 92.2%, 97.1% and 93.4%, respectively ; and all of 12 benign pNETs showed SUVmax below this cut-off value, although the case number is limited.Since the cut-off value to differentiate ductal cancer from mass-forming chronic pancreatitis was the same as the cut-off value to differentiate IPMC from benign IPMN that is 2.5), we unified the data of all malignant tumors and all benign tumors including pNETs—pNETs were not suitable for independent analysis due to their small number of cases—and re-computed a cut-off value to discriminate between malignant and benign tumors of the pancreas. The SUV5 Figure b. The ar.5, we unmax offering a semi-quantitative analysis is more advantageous. In this study, the cut-off value of SUVmax for differentiating pancreatic ductal cancer from mass-forming chronic pancreatitis was 2.5. This value was comparable with those obtained in other studies  because there was no difference between these 4 tumors and others concerning the tumor size and histological differentiation. Four of 18 patients (22.2%) having IPMC showed false-negative . These tumors were all CIS. To detect the CIS preoperatively is extremely difficult, and FDG-PET would regard CIS as benign lesion. There was only one false-positive lesion in mass-forming chronic pancreatitis. This case had IgG4-related AIP. If serum IgG4 level or other markers that indicate autoimmunity had been elevated preoperatively, we could suspect IgG4-related AIP in the first place. However, in our patient, these markers were all within normal limits. In this way, there are patients showing that serum IgG4 and other markers are within normal limits. In these cases, preoperative diagnosis is not easy. As to FDG-PET for IgG4-related AIP, some case reports are published, but the study of FDG-PET in differential diagnosis of IgG4-related AIP has not been performed yet because patient number is extremely limited for submitting analysis. Kamisawa et al. compared the SUVmax of autoimmune pancreatitis and pancreatic cancer  and one false-positive lesion in mass-forming chronic pancreatitis in detail. We cannot explain the reason why 4 patients (5.0%) having ductal cancer showed false-negative  were diagnosed as having pancreatic tumors by US, CT, and magnetic resonance imaging (MRI) in our department. All of them underwent FDG-PET. The diagnosis was histologically proven and the final diagnoses were 80 pancreatic ductal cancers, 10 mass-forming chronic pancreatitis, 18 intraductal papillary mucinous carcinoma (IPMC), 12 benign IPMN, 4 malignant pNETs (non-functioning islet cell carcinoma) and 12 benign pNETs . The research protocol of this study was approved by the Ethics Committee of Akita University Graduate School of Medicine (No.1223), and each patient gave written informed consent before enrollment in the study.18F-FDG by the tumors is reduced in hyperglycemic status, the guideline for FDG-PET/CT recommends that examination be performed under the blood glucose level lower than 150-200 mg/dl  with 10 subsets and 2 iterations. A region of interest (ROI) was designated at the site of maximal accumulation within the mass lesion. The maximal radioactivity of the ROI was determined. FDG uptake was calculated as SUV according to the following formula  = 7.40 × 106):FDG-PET studies were performed either with Headtome V  or with PET-CT scanner of Discovery ST Elite 16 . Patients having pancreatic cancer and/or chronic pancreatitis often show hyperglycemia. Because the uptake of max between the groups was performed by Mann-Whitney U test. A P value of < 0.05 was considered statistically significant. The SUVmax threshold  was determined by the receiver operating characteristic (ROC) analysis. ROC analysis was performed using calculation software IBM SPSS. The performance of the ROC analysis was verified by the ROC-area index Az. The cut-off point was determined by the Youden Index.The data presented were expressed as means ± standard deviation (S.D.) The statistical analysis of SUV


Considering nearly 40% of multidomain proteins contain one or more discontinuous domains, we have developed DomEx to enable domain boundary predictors to detect discontinuous domains by assembling the continuous domain segments. Discontinuous domains are predicted by matching the sequence profile of concatenated continuous domain segments with the profiles from a single-domain library derived from SCOP and CATH, and Pfam. Then the matches are filtered by similarity to library templates, a symmetric index score and a profile-profile alignment score. DomEx recalled 32.3% discontinuous domains with 86.5% precision when tested on 97 non-homologous protein chains containing 58 continuous and 99 discontinuous domains, in which the predicted domain segments are within ±20 residues of the boundary definitions in CATH 3.5. Compared with our recently developed predictor, ThreaDom, which is the state-of-the-art tool to detect discontinuous-domains, DomEx recalled 26.7% discontinuous domains with 72.7% precision in a benchmark with 29 discontinuous-domain chains, where ThreaDom failed to predict any discontinuous domains. Furthermore, combined with ThreaDom, the method ranked number one among 10 predictors. The source code and datasets are available at  Proteins consist of one or several stable, compact, and autonomously folding substructures, which are referred to as domains. The identification of protein domains plays an important role in determining protein structures by experimental methods including Nuclear Magnetic Resonance (NMR) and X-ray crystallography,2. MeanwAlthough protein domains usually have a single continuous segment of protein chain, there are still many domains formed from two or more nonsequential segments, which are called “discontinuous domains”. For exaOver the last three decades, a number of methods have been developed to identify protein domains, which are roughly classified into two categories according to their input data: structure or sequence. The structure-based methods can accurately identify continuous and discontinuous domains from the atomic coordinates of proteins ,11–15. TAn accurate discontinuous domain prediction includes predicting the accurate domain boundaries and the number of segments within one discontinuous domain. Currently, the sequence-based methods mainly focused on domain number and boundary prediction. DGS guesses Some methods such as SnapDRAGON, RosettaIn this work, we present a new strategy, DomEx, to enable continuous domain boundary predictors to predict discontinuous domains based on the sequence segment assembly. A template similarity score, symmetric index score and a profile-profile alignment score were developed to detect the discontinuous domains through a comprehensive single-domain library collecting not only from the structure domain databases  from N- to C-terminal, and it has a homologous domain partner B. The position of the last residue of segment A1 is marked as n, then the alignment pair (A-B) could be divided into two segment pairs (A1-B1 and A2-B2) at the position between n and n+1. The coverage and sequence similarity of the segment pair A1-B1 should be close to that of the segment pair A2-B2. In other words, the coverage and sequence similarity between the two segment pairs are symmetric at the separated point.DomEx makes three assumptions: (a) Homologous protein domains can be detected by sequence-based profile-profile alignments; (b) Homologous domain pairs have approximately similar length; (c) The coverage and sequence similarity between the different segment pairs in the same homologous domain pairs are usually symmetric. Assumptions (a) and (b) are straightforward. For assumption (c), let some discontinuous domain A has two segments  score.Step 6: Detect conflicts and report the final result.The entire flowchart of DomEx is shown in Qi is predicted as discontinuous, if there is at least one hit Tj with length error e< 0.2, Ts-score>TTS and SI<TSI. The parameters TTS and TSI are the cutoffs of TS-score and Symmetric Index, and they satisfy the constraint functionTSI = f. This function can be decided by maximizing the Matthews Correlation Coefficient (MCC) value in the training datasets (see below).An assembled domain sequence If there are multiple candidates that pass through the decision tree, then the candidate with the lowest PPA-score is selected.X which is divided into n+1 segments  by n boundary bars , which can be predicted by ThreaDom  and , respectively. The average MCC, recall and precision for the training dataset with different TTS and TSI are shown in An exhaustive grid search of the 2-dimensional space of TS-score and SI was performed to find the best combination of cutoffs The Symmetric Index becomes more important when TS-score <0.5. The highest MCC values (> 0.3) are in the region of TS-score ∈ and SI∈, where DomEx has a reasonable precision (>0.6) and maximum recall (>0.34).TSI = f with a dynamic relationship between the cutoff TTS and the cutoff TSI controlled by a single parameter b, i.e.TTS = 0.30, and we get TSI = 0.2 according to the TSI = 0.30 and SI≤TSI = 0.2. Algorithm 2 . The PPA-score gives the alignment quality between the query and template sequence. The score is usually negative. A low score means a good alignment. In the ECV training, there are 109 out of the 288 positive samples correctly detected as discontinuous, and 72 out of the 548 negative samples incorrectly detected as discontinuous when b>0.1. We use a total of 181 alignments as input to train the best PPA-score cutoff. We found the PPA can improve the MCC and precision when b<0.5 and PPA-score <-1.90 when the templates are collected from Pfam. Then the query is treated as a “Positive” detection, when the parameter b<0.5 and the PPA-score <-1.90.TPPA<−1.90 as the default parameters in DomEx.We tested the method on 390 discontinuous-domain samples on the Validation Dataset, using b from 0.9 to 0.1 with step -0.1 and PPA-score <-1.90 when b<0.5. Similar to the tendency on the Training Dataset, the precision of DomEx in the Validation Dataset varies from 1.0 to 0.771. The MCC values are all higher than 0.30 with recall ranging from 0.183 to 0.339. For better balance between precision and recall, we selected b = 0.3 and PPA-score cutoff To detect the discontinuous domains, DomEx depends on the predicted boundaries from other predictors, such as ThreaDom. The test dataset TEST-SET-I contains 99 positive and 58 negative discontinuous domains which were derived from a non-redundant set of protein chains consisting of 97 discontinuous domains . Each boundary was predicted by ThreaDom within an error of ±20 residues to the boundary definitions in CATH 3.5.In As a control, we employed five publicly available domain predictors, including ThreaDom, FIEFDomTo test the situation where there are only weakly homologous templates, for ThreaDom, we excluded all templates that have a sequence identity >30% to the target protein, and we also excluded templates that are detectable by PSI-BLAST with an E-value<0.05.For FIEFDom, we kept two group boundary prediction results, FIEFDom I and FIEFDom II. FIEFDom I excludes the templates if their sequence identity >30%; while FIEFDom II includes all the templates.The dataset TEST-SET-II contains 481 multi-domain chains, which is the same size as the training and validation sets. It includes 326 discontinuous chains and 155 continuous domain chains. Each chain has at least 3 segments, and the length of each segment is not less than 40 residues. When detecting the discontinuous domains, both DomEx with ThreaDom boundaries (ThrDm_Bdr+ DomEx) and DomEx with FIEFDom II boundaries (FIEFDom II+DomEx) have a higher NDO-score than their boundary prediction without DomEx detection. And the NDO-score of ThrDm_Bdr+DomEx (0.70) is higher than all the predictors that do not support discontinuous-domain detection. ThreaDom+DomEx has the highest NDO-score of 0.759. The results demonstrate that DomEx can improve the discontinuous domain detection when combined with other boundary predictors because of the addition of a sequence-based domain library and the symmetric alignment score.There are a total of 17 targets which have > = 3 continuous segments (length > 30 residues) from CASP8 to CASP10 experiments. Six contain multiple continuous domains, and eleven contain at least a discontinuous domain. A summary of the domain definitions from the CASP assessors is listed in b>0.8), DomEx achieves a high precision and is independent of the template sources (CATH+SCOP or Pfam). When b<0.8, the accuracy of the Pfam-based prediction is significantly lower than CATH+SCOP. If the template comes from CATH or SCOP, DomEx has a high precision (>90%). If the templates come from Pfam, the precision declines to about 50% when b is less than 0.65.The domain sequence library of DomEx is based on CATH, SCOP and Pfam-A. The domain boundaries of SCOP and CATH are defined based on the 3D structure of the proteins. Pfam-A is based on the HMM classification of sequences from whole-genomes. It is observed that the accuracy of templates from the Pfam library is about 60~70% of that from CATH+SCOP. b. For large b, the templates come mainly from CATH and SCOP, while for small b, much more templates are from Pfam.  When b = 0.2, about 70% of the templates come from Pfam, and the recall of DomEx is about 44%. The recall is 100% higher than when b = 0.5, where 50% of the templates come from Pfam. The templates from Pfam increase the recall of DomEx, even though the precision of DomEx decreases when b decreases. The domain sequences from Pfam improve recall because of its large size relative to the other databases.TPPA in b when TPPA varies from 0.0 to -5.0 with step -0.1. The average MCC and precision of Group I, which are 0.341 and 0.901, respectively, are higher than the other two. From the table, one can see that Group I has higher recall and MCC values for the cases when the precision values are similar to the other groups (underlined in table). For example, Group I has precision of 80.6% at b = 0.3, which is a bit higher than Group II at b = 0.4 and Group I at b = 0.5, while its recall is 31% and 61% higher than the other two, respectively.In DomEx, the Profile-Profile Alignment combined with the predicted secondary structure information is used to help DomEx improve the low precision when the templates are from Pfam. PPA filtering is not as effective for Group II as it is for Group I. The main reason is the domain definitions from SCOP and CATH are more likely to be correct than Pfam since they are derived from known 3D structures. Therefore, we used PPA filtering only for templates from Pfam library.In the segment assembly, we considered only the segment orderings from N- to C-termini, e.g. (A1A2) in Nevertheless, the reversed segment assembly can detect some interesting domain structures. All the three reversed cases are from proteins with segment-swapping domains (SSDs) . There aWe have proposed a new strategy, DomEx, to extend the ability of domain boundary predictors to detect discontinuous domains. The method assembles and detects discontinuous domains from the sequence segments. DomEx incorporates template similarity, symmetry of segment pairs, profile-profile alignments, and structure-based and structure-free libraries.https://github.com/xuezhidong/DomEx.Two test benchmarks showed that DomEx not only worked with the boundary predictors, but also was complementary to the discontinuous-domain detection method in ThreaDom. DomEx recalled 26.7% of the cases where ThreaDom failed. Half of these cases are attributed to templates from Pfam. When compared to other methods, DomEx plus ThreaDom gave the best NDO score, which further confirms that DomEx can detect discontinuous domains even without known 3D-structure templates. The main advantage of DomEx is that it searches for templates using domain-domain alignments rather than chain-chain alignments. Using domain-domain alignments improves recall because chain-chain alignments may miss templates where the domains match, but the rest of the chain does not match well. The benchmark results show that DomEx is an effective method, which opens the possibility of finding discontinuous domains in genome-wide studies. Currently, DomEx supports the detection of two-segment discontinuous domains. Further work will extend the model to detect discontinuous domains with more than 2 segments, and try to utilize the domain annotated information from CATH, SCOP and Pfam to enhance the performance of detecting discontinuous domains. The accuracy of boundary predictors and sequence alignment tools should also improve the detection results. The source code and datasets of DomEx are available at S1 Table(PDF)Click here for additional data file.


Besides, we found that the frequency of M-MDSCs in HCV infected patients had certain relevance with age ; patients older than 40 years old group  had a significantly higher frequency of M-MDSCs than that of age less than 40 years old group  . The frequency of M-MDSCs, however, had no correlation with HCV RNA loads, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and the level of liver inflammation degree.Myeloid-derived suppressor cells (MDSCs) have been shown to inhibit T-cell responses in many diseases, but, in hepatitis C virus (HCV) infected patients, MDSCs are still poorly studied. In this assay, we investigated the phenotype and frequency of two new populations of MDSCs denoted as monocytic and granulocytic MDSCs (M-MDSCs and G-MDSCs) in HCV infected patients and analyzed their clinical significance in these patients respectively. We found that the frequency of CD14 Several studies have shown that persistent HCV infection, which leads to the development of chronic hepatitis C (CHC) or hepatocellular carcinoma (HCC), was associated with impaired T cell responses. It is widely accepted that host immune injury, particularly the impaired T cell responses, plays an important role in HCV persistent infection –3. It haA total of 68 treatment-naive patients with HCV were enrolled from the Third Affiliated Hospital of Sun Yat-Sen University  from April 2012 to July 2010. The population recruited in this study was composed of three groups of subjects, including 56 CHC patients and 12 patients of hepatitis C related liver cirrhosis; 15 healthy controls were randomly selected from the medical center of the Third Affiliated Hospital of Sun Yat-Sen University. All the detailed characteristics of study subjects are presented in The study protocol was approved by the Ethics Review Board of the Third Affiliated Hospital of Sun Yat-Sen University. Written informed consent was obtained from the patients or their families prior to enrollment.Peripheral blood was drawn (10 mL) into EDTA anticoagulation tubes  from healthy controls and patients with HCV. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Amersham Biosciences) density gradient centrifugation within 4 hours. Cells were washed in RPMI 1640 media  twice and then resuspended in freeze medium (90% FBS (Life Technologies) and 10% DMSO ). Finally, PBMCs were transferred into cryovials , cryopreserved at −80°C, and 72 hours later transferred to the liquid nitrogen.6 cells/mL by complete medium.For analysis, cryovials were removed from the liquid nitrogen, and then were put into the 37°C water bath thawing quickly within 1 min. Then, the cells were resuspended in 10 mL of complete medium . After being washed twice, the cells were counted by a light microscope after trypan blue dye staining, and then we resuspended the cells and adjusted the concentration to 1 × 10+HLA-DR−/low (M-MDSCs) and HLA-DR−/lowCD33+CD11b+CD15+ (G-MDSCs) cells in PBMCs, the following labeled multicolor fluorescence anti-human monoclonal antibodies (mAbs) were used for surface staining: anti-HLA-DR-FITC, anti-CD33-APC, anti-CD11b-PE, anti-CD15-PERCP-Cy5.5, and anti-CD14-PERCP-Cy5.5 . The cells of each sample were incubated with these labeled multicolor fluorescence anti-human monoclonal antibodies at 4°C for 30 minutes. After surface staining, the cells were washed with 2 mL flow staining buffer (PBS plus 1% FBS) at 4°C and centrifuged at 400 g for 5 minutes. Cell pellets were diluted in 300 μL PBS after the supernatant was removed. Flow cytometry was done on a FACSCalibur. All FACS-data was analyzed by FlowJo software . Isotype-matched control antibodies were used as negative controls.To determine the frequency and phenotype of CD14Plasma HCV RNA was quantified by reverse transcriptase polymerase chain reaction assay . AST and ALT were tested in the laboratory center of our hospital with Hitachi7170 automatic biochemistry analyzer.t test of means between groups. For nonnormally distributed data, variables are expressed as medians, interquartile range, and the nonparametric Mann-Whitney U tests were used to compare differences between groups. The correlation between the frequency of MDSCs and patients clinical parameters was analyzed by Spearman's rank tests, and P value less than 0.05 was considered to be statistically significant.All data were analyzed by SPSS Statistics 17.0 and all figures were made by Prizm 5.0 statistical analysis software (GraphPad Software). Quantitative data normally distributed are expressed as mean ± standard, followed by the P = 0.015) (P > 0.05) (P < 0.001)  , while t > 0.05) . What is< 0.001) .P = 0.094) (P = 0.579)  . However= 0.579) .r = 0.358, P = 0.003)  (P = 0.007)      compared= 0.419) . Then we= 0.007) . In addi= 0.756) . Also, N= 0.069)  or ALT (= 0.279) .MDSCs were involved in the immune tolerance of various diseases, such as cancers, autoimmune diseases, and acute and chronic infection diseases –15, and β secretion [β and had nothing to do with the activity of arginase [In tumors, two main subsets of MDSCs were identified. One was referred to as M-MDSCs with the expression of CD14, which have a common phenotype and morphology but distinct function compared to monocytes. The other was G-MDSCs with the expression of CD15, which have common phenotype and significant suppressive activity compared to granulocytes , 17. Intarginase .−/lowCD33+CD11b+CD15+ MDSCs [In this study, we observed a significant elevation of M-MDSCs in peripheral blood of HCV infected patients compared to healthy controls. What is more is that the frequency of M-MDSCs was also higher than G-MDSCs in HCV infected patients, while the expression of G-MDSCs in HCV patients was as low as healthy controls. These results suggested that HCV infection could cause the proliferation of M-MDSCs. As we all know the density gradient centrifugation has been a traditional method to separate PBMCs, such as lymphocytes and monocytes, from polymorphonuclear cells. Most granulocytes, mature CD15 positive cells, deposit to the cell pellet after Ficoll centrifugation because of their high density. Nevertheless, it is possible that some granulocytes with slightly lower density persisted and accounted for the HLA-DR5+ MDSCs –28. Besi5+ MDSCs , which wHCV is prone to cause persistent infection and result in HCV related cirrhosis. In addition to the virus mutation, the impaired immune system plays a main role in liver damage. The frequency of M-MDSCs or G-MDSCs in CHC patients was nearly equivalent to CHC patients with liver cirrhosis in this study, which might indicate that these two MDSCs subsets make little sense to the disease progression from chronic CHC to cirrhosis. What is more is that we found that the frequency of M-MDSCs was associated with the age of patients compared to healthy volunteers. Then we divided the HCV patients into age > 40 yr group and age ≤ 40 yr group and found that M-MDSCs in the former group was significantly higher than that of the latter group. It may be related to the duration of HCV infection. As it has found that the younger age of the patients, the shorter duration they may be infected. In other studies, It has been reported that the frequency of M-MDSCs is correlated with the clinical biochemical parameters of HCV patients, including HCV viral load and the level of ALT and AST which reflect liver injury , 30. HowWith the complexity and heterogeneity of MDSCs, it is difficult to accurately analyze the phenotype of MDSCs subpopulations in human and their clinical significance. Here we have demonstrated a significant accumulation of M-MDSCs rather than G-MDSCs in peripheral blood of HCV infected patients and we also explored the clinical significance of M-MDSCs and G-MDSCs in HCV infected patients. This observation may help to shed light on the possible role of M-MDSCs in HCV infected patients and lay a foundation for the immune targeted therapy to hepatitis C.


Ceria nanoparticles (nano-ceria) have recently gained a wide range of applications, which might pose unwanted risks to both the environment and human health. The greatest potential for the environmental discharge of nano-ceria appears to be in their use as a diesel fuel additive. The present study was designed to explore the pulmonary toxicity of nano-ceria in mice after a single exposure via intratracheal instillation. Two types of nano-ceria with the same distribution of a primary size (3–5 nm), but different redox activity, were used: Ceria-p, synthesized by a precipitation route, and Ceria-h, synthesized by a hydrothermal route. Both Ceria-p and Ceria-h induced oxidative stress, inflammatory responses and cytotoxicity in mice, but their toxicological profiles were quite different. The mean size of Ceria-p agglomerates was much smaller compared to Ceria-h, thereby causing a more potent acute inflammation, due to their higher number concentration of agglomerates and higher deposition rate in the deep lung. Ceria-h had a higher reactivity to catalyzing the generation of reactive oxygen species (ROS), and caused two waves of lung injury: bronchoalveolar lavage (BAL) inflammation and cytotoxicity in the early stage and redox-activity-evoked lipid peroxidation and pro-inflammation in the latter stage. Therefore, the size distribution of ceria-containing agglomerates in the exhaust, as well as their surface chemistry are essential characteristics to assess the potential risks of using nano-ceria as a fuel additive. The widet al. predicted that receptors in the car passenger area of a street canyon would be exposed to ceria at a concentration up to 80 ng/m3 in the worst case [Nano-ceria are used increasingly as an additive to improve the burning efficiency of fuels, reducing fuel consumption, greenhouse gases and particle numbers in vehicle exhaust . Nanoscarst case . The comrst case . FurtherHowever, the physicochemical properties of nano-ceria would be changed during combustion, and the details of the changes remain unknown. For example, the reported crystallite size of nano-ceria in diesel exhaust were quite controversial: ranging from 1–3 nm , 5–7 nm Recent literature has shown that nano-ceria exposure is associated with pulmonary injury, leading to oxidative stress, inflammation and cytotoxicity –11. In oThe size of ENMs plays a key role in determining the toxicity of ENMs . Compare2.2.1.vs. 1731 ± 165 nm. Therefore, particle agglomeration would occur after Ceria-p and Ceria-h were instilled into the airway, and Ceria-h agglomerates had a significantly larger size distribution compared to Ceria-p agglomerates. X-ray absorption near-edge structure (XANES) and X-ray diffraction (XRD) measurement demonstrated that Ceria-p and Ceria-h were the same in the oxidation state of cerium and in the crystalline peaks (3+ (The two types of nano-ceria were synthesized via precipitation (Ceria-p) or the hydrothermal (Ceria-h) method, respectively. The morphologies of Ceria-p and Ceria-h were studied by transmission electron microscopy (TEM), and the size distributions of their primary particles were very similar, within a range of 3–5 nm . Howeverne peaks . In the eaks . In spite of the enhanced anti-oxidative capacity, Ceria-h exposure resulted in oxidative damage at 7 and 28 days PI, implying an unbalance between the oxidative stress and anti-oxidative defenses.2.4.The histopathological changes in lung were evaluated with hematoxylin-eosin (HE) staining in 3.et al., the commercial use of nano-ceria will increase the air-borne nano-ceria, to a level of 80 ng/m3 in the worst case [Nano-ceria have recently gained a wide range of applications, which might pose unwanted risks to both the environment and human health. The greatest potential for the environmental discharge of nano-ceria appears to be in their use as a diesel fuel additive. According to the prediction by Park rst case . In the Although the toxic mechanisms for inhaled ENMs were not very clear, the results of older toxicological and epidemiological studies with airborne fine or ultrafine particles can be viewed as the basis for the expanding field of nanotoxicology . Due to et al. reviewed the reports focusing on particle-overload in lung and concluded that a dose that can induce inflammation is between one and 5 mg/kg [Studies in rodents imply that the excessive burden of insoluble particles of low cytotoxicity would lead to pulmonary inflammation . Oyabu e 5 mg/kg . Our preHowever, there were some differences between the toxicological profiles of Ceria-p and Ceria-h. In general, intratracheal instillation of Ceria-p induced transient inflammatory responses in both BAL cells and lung tissue at the early stage of exposure (one and/or three days PI), which recovered at 7 or 28 days PI. Ceria-h induced initially minor, but more persistent inflammation in BAL cells and delayed lipid peroxidation and pro-inflammation in lung tissue at 28 days PI.et al. found that the pulmonary toxicity of ENMs might depend not solely on the dimension of the ENMs, but also on the dimension of the ENMs agglomerates [2 agglomerates (>100 nm) caused acute inflammatory responses; small ones (<100 nm) exerted both cytotoxicity and oxidative stress without apparent neutrophil influx into the airways, because agglomerates smaller than 100 nm could easily escape the pulmonary clearance via phagocytosis [The differences in the initial inflammatory responses may be due to the different agglomeration state of Ceria-p and Ceria-h. The size of agglomerates would determine their deposition in the lung, their ability to cross biological barriers and their cellular internalization . Noël etomerates . Howeverocytosis ,17. In tIn the lungs instilled with Ceria-h, oxidative damage was found at seven and 28 days PI, and a pro-inflammatory effect was found at 28 days PI. These delayed responses seemed not to be attributed exclusively to the excessive ENMs burdens in the lung, as particle-laden macrophages were less observed in the Ceria-h treated mice than in the Ceria-p group. ROS generation is identified as one of the major mechanisms by which inhaled ENMs exert adverse biological effects ,19. We s3+ and Ce4+ makes nano-ceria attractive as a fuel additive to reduce the release of greenhouse gases and particles. Meanwhile, nano-ceria are being proven promising for their SOD mimetic activity [3+/Ce4+ ratio on the surface could exhibit SOD mimetic activity, whereas nano-ceria with a lower Ce3+/Ce4+ ratio show no SOD mimetic activity [3+ 3 was added to NaOH solution (0.4 g NaOH dissolved in 128 mL water) followed by 48 h of magnetic stirring. The resulting white precipitate was collected and washed several times in ultrapure water.The primary particle diameters of the two types of nano-ceria used in the present work were both 3–5 nm. One of the nano-ceria (Ceria-p) was synthesized by a precipitation method . In brie3)3 solution was mixed with 15 mL toluene; then, 22.5 mL of 233.3 mmol/L sodium oleate aqueous was dropped into the above mixture solution with magnetic stirring. The upper layer toluene with cerium precursor was transferred to a 50-mL Teflon-lined stainless-steel autoclave with 15 mL deionized water and 0.35 mL tert-butylamine. The sealed autoclave was transferred to a 180 °C oven, held there for 12 h and then cooled to room temperature. Then, the brown supernatant solution with synthesized nano-ceria was precipitated with an adequate volume of ethanol. The precipitate was obtained by centrifugation for 10 min and re-dispersed in 15 mL ultrapure water containing 400 μL of 3-mercaptopropionic acid (3-MPA). After centrifuging and washing 3 times, a Ceria-p suspension was obtained.Another type of nano-ceria (Ceria-h) was synthesized by a surfactant-assisted hydrothermal approach . Briefly+#, which was measured at 405 nm by using a UV-vis spectrophotometer . Reactions with nano-ceria were buffered by Tris . The solution containing 100 μM nano-CeO2, 88 mM H2O2 and 100 μM ABTS was added into a 1-cm path length quartz cuvette, and the absorbance at 405 nm was recorded once every 3 s for 10 min.The physicochemical properties of nano-ceria were characterized by transmission electron microscopy , dynamic light scattering , X-ray absorption near-edge structure , and X-ray diffraction . The ROS generation catalyzed by nano-ceria was determined by the method described by the literature of Heckert , using 24.2.ad libitum. After one week of acclimation, mice were randomly divided into 12 groups, with 8 mice per group and housed individually in stainless steel cages under standard laboratory conditions . All the animal experiments were performed with the approval of the Ethics Committee of Animal Care and Experimentation of the National Institute for Environmental Studies, China.Male CD-1 (ICR) mice (6 weeks old) were purchased from Beijing Vital River Laboratories. A commercial pellet diet and deionized water were available 4.3.The Ceria-p and Ceria-h were dispersed in ultrapure water at a concentration of 0.8 mg/mL, respectively, and sonicated for 15 min before use. By using a non-surgical intratracheal instillation method [4.4.g and 4 °C for 10 min. The supernatant was used for biochemical analyses, while the recovered cells from the pellet were resuspended for the macrophage, neutrophil and lymphocyte cell counting with Wright’s staining. The activities of lactate dehydrogenase (LDH) and acid phosphatase (ACP) in the BALF were analyzed using colorimetric assay kits . The concentration of total protein (TP) was determined by the bicinchoninic acid (BCA) method using a BCA protein assay kit  with bovine serum albumin as a standard.The animals were sacrificed at 1, 3, 7 and 28 days post-instillation (PI). Bronchoalveolar lavage was performed 3 times with sodium phosphate buffer , and the cells in BALF were collected by centrifugation at 400× 4.5.v/v = 1/4). The homogenates were centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatants were aliquoted and stored at −80 °C for the biochemical analysis. The concentrations of malondialdehyde (MDA) and GSH, as well as the activities of SOD, GSH-Px and CAT in the lung homogenates were measured using the reagent kits purchased from Jiancheng Bioengineering Co., Ltd., Nanjing, China, according to the manufacturer’s protocols. Protein levels in the lung homogenates were determined by the bicinchoninic acid (BCA) protein assay kit . The levels of IL-6 in the lung homogenates were determined using commercially available ELISA kits .The lung tissues were separated and homogenized by a glass homogenizer in pre-cooled 0.01 M PBS .4.7.*p < 0.05 was considered statistically significant.All data were expressed as the mean ± standard deviation (SD). Statistical analyses were conducted with SPSS 16.0  statistical software for Windows. A one-way analysis of variance (ANOVA) and Tukey’s test were carried out to illustrate the significant difference between the treatment, and a difference of 5.In the present work, we investigated the pulmonary toxicities of two kinds of nano-ceria with the same primary size distribution. Both Ceria-p and Ceria-h induced oxidative stress, inflammatory responses and cytotoxicities in mice, but possibly via different mechanisms. The mean size of Ceria-p agglomerates was much smaller compared to Ceria-h, thereby causing more potent, acute inflammation, due to its higher concentration number of agglomerates and higher deposition rate deep in the lung. Ceria-h exposure caused two waves of lung injury: BAL inflammation and cytotoxicity in the early stage and redox-activity-evoked lipid peroxidation, as well as pro-inflammation in the latter stage. Therefore, agglomerates size, as well as the surface chemistry of the combusted nano-ceria in the exhaust are essential characteristics to assess the potential risks of using nano-ceria as a fuel additive.


Its concentration was highly heterogeneous among OvCa patients, without any correlations with the FIGO stage and the pre-treatment CA-125 level. The decrease in CA-125 concentration was constant and often dramatic, while the variations of miR-200b concentration were much more diverse. The variation of miR-200b was marginally associated with the PFS  while miR-200b as a continuous time-dependent variable was significantly associated . This study is the first direct empirical evidence that miR-200b can provide additional information, independent of CA-125 in OvCa patients.Ovarian carcinomas (OvCa) are highly heterogeneous malignancies. We investigated four circulating plasma microRNAs  as candidate biomarkers. Using qPCR, we assessed the plasma concentration of these markers in 101 women, including 51 previously untreated OvCa patients, 25 healthy women and 25 patients bearing benign pelvic lesions. For a subset of 33 OvCa patients, the assay was repeated at the end of the primary treatment. The pattern of variations (post- minus pre-treatment) of concentration was compared to that of CA-125. A Cox regression model was used to study the association between variations and the progression-free survival (PFS). Plasma miR-200b proved to have a greater average concentration in OvCa samples (median 2 In developed countries, ovarian carcinoma (OvCa) is the most lethal gynecologic cancer. High-grade serous ovarian carcinoma (HGSOC) is the most frequent (60%) and aggressive type of ovarian malignancies , 2. Its Most ovarian carcinomas are found in stage III (extension to the peritoneum and/or lymph nodes) or IV, with a five-year survival rate inferior to 30% . SurgicaMicroRNAs are small, single-stranded non-coding RNAs, about 19-25 nt long, which play key roles in the regulation of gene expression at the post-transcriptional level . They arOur initial aim was the reproduction of previous findings on 4 circulating miRNAs in 51 women hospitalized for primary ovarian cancer. High concentrations in plasma samples from OvCa were found only for miR-200b. In contrast, the distribution of the 3 other selected miRNAs was highly similar in samples from untreated patients bearing OvCa or benign tumors. A longitudinal study (pre/post-treatment) was conducted, assessing the plasma levels of miR-200b. Our findings show that circulating miR-200b can provide additional information to that provided by CA-125. In contrast to CA-125, variations of miR-200b (post- minus pre-treatment) are marginally significantly associated with progression-free survival (PFS).N = 4), 3.9% stage II (N = 2), 82.4% stage III (N = 42) and 5.9% stage IV (N = 3). The 2 control groups were 25 HW and 25 BT . The mean age was 62 years  with 7.8% FIGO stage I  in OvCa compared to 1.26  in BT  suspected to have a frequent high concentration in plasma samples from OvCa patients compared to BT patients, only miR-200b was found with a distinct pattern of distribution in these patients. The concentration median 22 = 0.0974, p = 0.01), which was not observed for miR-200b (p = 0.93). Regarding the four cases of early disease (stages Ia-Ic), CA-125 was very low in all of them whereas the concentration of miR-200b was above its average concentration for three of them.As shown in Figure For 33 of the 51 OvCa patients, we were able to collect a second blood sample, at the end of their primary treatment (sample B in addition to the previously mentioned sample A collected prior to any treatment). The variations of CA-125 and miR-200b were designated ΔCA-125 and ΔmiR-200b (concentration of sample B minus concentration of sample A for CA-125 and miR-200b respectively). In almost every case, CA-125 was returning to normal plasma concentrations within the first months of the treatment, even among patients of category 1 (bearing unresectable tumors). At the end of the treatment, CA-125 was either normal (24 patients) or slightly above the limit (5 patients). This is illustrated in Figure p-value = 0.018).To investigate the association between ΔCA-125 ( < or ≥ 0) and ΔmiR-200b ( < or ≥ 0) with the PFS, a landmark analysis at 10 months was used. This time space represents the highest time interval between evaluation of sample A and sample B. We imputed, for patients without evaluation of sample B at 10 months, the value before this time. Nine patients with an event recorded within the first 10 months had to be excluded from this analysis. Figure p = 0.03), and 2.33,  (p = 0.11) to the detriment of ΔmiR-200b≥0. When also adjusting on the initial plasma concentration of miR-200b prior to any treatment as a continuous covariate, univariate and multivariable, HR for the variation were 3.14  (p = 0.06) and 2.95  (p = 0.06), respectively. In other words, the risk of progression was marginally significantly higher in patients with a positive variation of miR-200b compared to patients with a negative variation. It is interesting to note that the initial value of plasma miR-200b was not associated with PFS (p = 0.24). The extension of the Cox regression model including miR-200b as a time-dependent covariate  showed that the association was now statistically significant . We recorded a 5.7% higher risk of progression for a 10 unit increase of miR-200b concentration. No significant association between CA-125 and PFS was observed when considering the initial value of the marker (p = 0.42) or when taking it as a time-dependent covariate (p = 0.40).In order to confirm this observation and to investigate whether the prognostic value of ΔmiR-200b was independent from some major clinical characteristics like tumor extension and the possibility of tumor resection, we used a multivariable analysis based on a Cox model adjusted on tumor stage  and primary treatment category . The univariate and multivariable hazard ratios (HR) were 3.12, 95% confidence interval =  . If the variation of miR-200b is validated by future studies as a novel biomarker, it will be a tool of major interest for the management of patients on remission assessed by imaging or second-look surgery. Determining the patients at high risk of relapse could lead to the tailoring of more efficient adjuvant therapy approaches. The confirmation of the predictive value of miR-200b will require the investigation of new series of OvCa patients with greater numbers of sequential samples through the primary treatment. This will allow a better assessment of the kinetics of variation for miR-200b.MiR-200b should not be regarded as a possible substitute of CA-125 but rather as a complementary marker. Two major advantages of CA-125 in the management of OvCa are its contribution to the initial diagnosis and the post-treatment surveillance. On the one hand, in the presence of a pelvic mass, the detection of the CA-125 strengthens the suspicion of ovarian carcinomas prior to laparoscopy or laparotomy . On the The miR-200 family contains five distinct members: miR-141, miR-200a, miR-200b, miR-200c and miR-429 -23, suggIt is puzzling to observe that plasma miR-200b can rise in some patients in parallel with a substantial reduction of the tumor mass or even a complete remission. The elucidation of this paradox will be important for its future use as a prognostic marker. At this stage, one can only speculate on the role of hidden tumor cells not easily seen by medical imaging or second-look surgery, or a change of phenotype in cells resistant to the chemotherapy and likely to be involved in tumor relapse.Blood samples were collected prospectively from female patients admitted at Gustave Roussy, with written informed consent under the agreement of the ethical committee . The blood (10-20 ml) was collected in EDTA tubes and kept at room temperature for less than 2 hours, until plasma separation. Subsequently, it was centrifuged at 1700 g at 20°C for 15 minutes to separate plasma from blood cells. The plasma was homogenized, aliquoted and stored at −80°C, for 6-36 months. As controls, we used 25 samples from healthy women (HW), proved to be free of BRCA1/BRCA2 mutations detected in their family. Besides, we used 2 sets of pathological samples: one obtained from 25 women operated for benign pelvic tumors or lesions (BT hereafter) and a second set obtained prior to any treatment, before a diagnostic coelioscopy, from 72 women treated for malignant ovarian tumors (samples A). Among them, 33 women donated a second sample (samples B) at the end of the primary treatment (treatment including chemotherapy and debulking surgery when feasible), 4-8 months after the initial coelioscopy. For all patients, the follow-up time was at least 6 months from the diagnosis.The extraction of all small RNAs was done using the miRCURY RNA isolation kit of Exiqon, optimized for miRNA extraction from biofluids. For each extraction we used 200 μL of undiluted plasma, supplemented with 1 μg of carrier RNA  per sample, in order to improve microRNA yield, according to the manufacturer's manual. The total RNA concentration was assessed using a Nanodrop2000 spectrophotometer.Prior to retrotranscription, the samples were diluted to a final concentration of 5 ng/μl, including the carrier RNA. To exclude immature forms of miRNAs, we used Exiqon's Universal cDNA Synthesis kit II, which adds a poly-A tail to the extracted RNAs before the cDNA synthesis. Two microliters of diluted microRNA extract were mixed with 5 μl of RNase-free water, 2 μl of 5X Reaction buffer and 1 μl of a 10X enzyme mix, containing the reverse transcriptase and a poly-T primer with a 3′ degenerate anchor and a 5′ universal tag. The tubes were incubated at 42°C for 1 hour and at 95°C for 5 minutes, in order to heat-inactivate the enzyme and then stored at −20°C immediately after.http://atlas.dmi.unict.it/mirandola/). Each of them has been reported in at least two publications as exhibiting a high plasma or serum concentration in OvCa patients  of the candidate microRNAs was compared between BT and OvCa using the Kruskal-Wallis test. We used the false discovery rate adjustment to control for multiple testing. We studied the correlation between the selected miRNA and CA-125 plasma concentrations through a linear regression and R2 after log transformation. The pre/post-treatment variations (noted Δ = sample B - sample A and defined as < or ≥0) of CA-125 and miRNA plasma concentrations were reported  by category of primary treatment and overall. Progression-free survival (PFS) was defined as the time from the start of the treatment to the first progression, as defined according to GCIG criteria [P-values were two-tailed. We used Graph Pad Prism 4 for the figures and SAS 9.3 for statistical analyses.Patients' characteristics and treatment modalities were retrieved from Gustave Roussy medical files. The distribution of the concentrations (2criteria . Patientcriteria . P-value